Short- and Long-Term Adaptation to Altered Levels of Glucose: Fifty Years of Scientific Adventure.

My graduate and postdoctoral training in metabolism and enzymology eventually led me to study the short- and long-term regulation of glucose and lipid metabolism. In the early phase of my career, my trainees and I identified, purified, and characterized a variety of phosphofructokinase enzymes from mammalian tissues. These studies led us to discover fructose 2,6-P2, the most potent activator of phosphofructokinase and glycolysis. The discovery of fructose 2,6-P2 led to the identification and characterization of the tissue-specific bifunctional enzyme 6-phosphofructo-2-kinase:fructose 2,6-bisphosphatase. We discovered a glucose signaling mechanism by which the liver maintains glucose homeostasis by regulating the activities of this bifunctional enzyme. With a rise in glucose, a signaling metabolite, xylulose 5-phosphate, triggers rapid activation of a specific protein phosphatase (PP2ABδC), which dephosphorylates the bifunctional enzyme, thereby increasing fructose 2,6-P2 levels and upregulating glycolysis. These endeavors paved the way for us to initiate the later phase of my career in which we discovered a new transcription factor termed the carbohydrate response element binding protein (ChREBP). Now ChREBP is recognized as the masterregulator controlling conversion of excess carbohydrates to storage of fat in the liver. ChREBP functions as a central metabolic coordinator that responds to nutrients independently of insulin. The ChREBP transcription factor facilitates metabolic adaptation to excess glucose, leading to obesity and its associated diseases.

The bifunctional 6-phosphofructokinase-2/fructose-2,6-bisphosphatase from the shrimp Litopenaeus vannamei: Molecular characterization and down-regulation of expression in response to severe hypoxia.

Hypoxia is a frequent stressor in marine environments with multiple adverse effects on marine species. The white shrimp Litopenaeus vannamei withstands hypoxic conditions by activating anaerobic metabolism with tissue-specific changes in glycolytic and gluconeogenic enzymes. In animal cells, glycolytic/gluconeogenic fluxes are highly controlled by the levels of fructose-2,6-bisphosphate (F-2,6-P2), a signal metabolite synthesized and degraded by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). PFK-2/FBPase-2 has been studied in vertebrates and some invertebrates, but as far as we know, there are no reports on PFK-2/FBPase-2 from crustaceans. In the present work, we obtained cDNA nucleotide sequences corresponding to two mRNAs for PFK-2/FBPase-2 and named them PFKFBP1 (1644 bp) and PFKFBP2 (1566 bp), from the white shrimp L. vannamei. The deduced PFKFBP1 and PFKFBP2 are 547 and 521 amino acids long, respectively. Both proteins share 99.23% of identity, and only differ in 26 additional amino acids present in the kinase domain of the PFKFBP1. The kinase and phosphatase domains are highly conserved in sequence and structure between both isoforms and other proteins from diverse taxa. Total expression of PFKFBP1-2 is tissue-specific, more abundant in gills than in hepatopancreas and undetectable in muscle. Moreover, severe hypoxia (1 mg/L of DO) decreased expression of PFKFBP1-2 in gills while anaerobic glycolysis was induced, as indicated by accumulation of cellular lactate. These results suggest that negative regulation of PFKFBP1-2 at expression level is necessary to set up anaerobic glycolysis in the cells during the response to hypoxia.

The phosphocarrier protein HPr of the bacterial phosphotransferase system globally regulates energy metabolism by directly interacting with multiple enzymes in Escherichia coli.

The histidine-phosphorylatable phosphocarrier protein (HPr) is an essential component of the sugar-transporting phosphotransferase system (PTS) in many bacteria. Recent interactome findings suggested that HPr interacts with several carbohydrate-metabolizing enzymes, but whether HPr plays a regulatory role was unclear. Here, we provide evidence that HPr interacts with a large number of proteins in Escherichia coli We demonstrate HPr-dependent allosteric regulation of the activities of pyruvate kinase (PykF, but not PykA), phosphofructokinase (PfkB, but not PfkA), glucosamine-6-phosphate deaminase (NagB), and adenylate kinase (Adk). HPr is either phosphorylated on a histidyl residue (HPr-P) or non-phosphorylated (HPr). PykF is activated only by non-phosphorylated HPr, which decreases the PykF Khalf for phosphoenolpyruvate by 10-fold (from 3.5 to 0.36 mm), thus influencing glycolysis. PfkB activation by HPr, but not by HPr-P, resulted from a decrease in the Khalf for fructose-6-P, which likely influences both gluconeogenesis and glycolysis. Moreover, NagB activation by HPr was important for the utilization of amino sugars, and allosteric inhibition of Adk activity by HPr-P, but not by HPr, allows HPr to regulate the cellular energy charge coordinately with glycolysis. These observations suggest that HPr serves as a directly interacting global regulator of carbon and energy metabolism and probably of other physiological processes in enteric bacteria.

Discover potential inhibitors for PFKFB3 using 3D-QSAR, virtual screening, molecular docking and molecular dynamics simulation.

The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) is a master regulator of glycolysis in cancer cells by synthesizing fructose-2,6-bisphosphate (F-2,6-BP), a potent allosteric activator of phosphofructokinase-1 (PFK-1), which is a rate-limiting enzyme of glycolysis. PFKFB3 is an attractive target for cancer treatment. It is valuable to discover promising inhibitors by using 3D-QSAR pharmacophore modeling, virtual screening, molecular docking and molecular dynamics simulation. Twenty molecules with known activity were used to build 3D-QSAR pharmacophore models. The best pharmacophore model was ADHR called Hypo1, which had the highest correlation value of 0.98 and the lowest RMSD of 0.82. Then, the Hypo1 was validated by cost value method, test set method and decoy set validation method. Next, the Hypo1 combined with Lipinski's rule of five and ADMET properties were employed to screen databases including Asinex and Specs, total of 1,048,159 molecules. The hits retrieved from screening were docked into protein by different procedures including HTVS, SP and XP. Finally, nine molecules were picked out as potential PFKFB3 inhibitors. The stability of PFKFB3-lead complexes was verified by 40 ns molecular dynamics simulation. The binding free energy and the energy contribution of per residue to the binding energy were calculated by MM-PBSA based on molecular dynamics simulation.

Crystal structure of heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB2) and the inhibitory influence of citrate on substrate binding.

The heart-specific isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB2) is an important regulator of glycolytic flux in cardiac cells. Here, we present the crystal structures of two PFKFB2 orthologues, human and bovine, at resolutions of 2.0 and 1.8 Å, respectively. Citrate, a TCA cycle intermediate and well-known inhibitor of PFKFB2, co-crystallized in the 2-kinase domains of both orthologues, occupying the fructose-6-phosphate binding-site and extending into the γ-phosphate binding pocket of ATP. This steric and electrostatic occlusion of the γ-phosphate site by citrate proved highly consequential to the binding of co-complexed ATP analogues. The bovine structure, which co-crystallized with ADP, closely resembled the overall structure of other PFKFB isoforms, with ADP mimicking the catalytic binding mode of ATP. The human structure, on the other hand, co-complexed with AMPPNP, which, unlike ADP, contains a γ-phosphate. The presence of this γ-phosphate made adoption of the catalytic ATP binding mode impossible for AMPPNP, forcing the analogue to bind atypically with concomitant conformational changes to the ATP binding-pocket. Inhibition kinetics were used to validate the structural observations, confirming citrate's inhibition mechanism as competitive for F6P and noncompetitive for ATP. Together, these structural and kinetic data establish a molecular basis for citrate's negative feed-back loop of the glycolytic pathway via PFKFB2. Proteins 2016; 85:117-124. © 2016 Wiley Periodicals, Inc.

The folding unit of phosphofructokinase-2 as defined by the biophysical properties of a monomeric mutant.

Escherichia coli phosphofructokinase-2 (Pfk-2) is an obligate homodimer that follows a highly cooperative three-state folding mechanism N2 ↔ 2I ↔ 2U. The strong coupling between dissociation and unfolding is a consequence of the structural features of its interface: a bimolecular domain formed by intertwining of the small domain of each subunit into a flattened ß-barrel. Although isolated monomers of E. coli Pfk-2 have been observed by modification of the environment (changes in temperature, addition of chaotropic agents), no isolated subunits in native conditions have been obtained. Based on in silico estimations of the change in free energy and the local energetic frustration upon binding, we engineered a single-point mutant to destabilize the interface of Pfk-2. This mutant, L93A, is an inactive monomer at protein concentrations below 30 µM, as determined by analytical ultracentrifugation, dynamic light scattering, size exclusion chromatography, small-angle x-ray scattering, and enzyme kinetics. Active dimer formation can be induced by increasing the protein concentration and by addition of its substrate fructose-6-phosphate. Chemical and thermal unfolding of the L93A monomer followed by circular dichroism and dynamic light scattering suggest that it unfolds noncooperatively and that the isolated subunit is partially unstructured and marginally stable. The detailed structural features of the L93A monomer and the F6P-induced dimer were ascertained by high-resolution hydrogen/deuterium exchange mass spectrometry. Our results show that the isolated subunit has overall higher solvent accessibility than the native dimer, with the exception of residues 240-309. These residues correspond to most of the ß-meander module and show the same extent of deuterium uptake as the native dimer. Our results support the idea that the hydrophobic core of the isolated monomer of Pfk-2 is solvent-penetrated in native conditions and that the ß-meander module is not affected by monomerizing mutations.

A fundamental trade-off in covalent switching and its circumvention by enzyme bifunctionality in glucose homeostasis.

Covalent modification provides a mechanism for modulating molecular state and regulating physiology. A cycle of competing enzymes that add and remove a single modification can act as a molecular switch between "on" and "off" and has been widely studied as a core motif in systems biology. Here, we exploit the recently developed "linear framework" for time scale separation to determine the general principles of such switches. These methods are not limited to Michaelis-Menten assumptions, and our conclusions hold for enzymes whose mechanisms may be arbitrarily complicated. We show that switching efficiency improves with increasing irreversibility of the enzymes and that the on/off transition occurs when the ratio of enzyme levels reaches a value that depends only on the rate constants. Fluctuations in enzyme levels, which habitually occur due to cellular heterogeneity, can cause flipping back and forth between on and off, leading to incoherent mosaic behavior in tissues, that worsens as switching becomes sharper. This trade-off can be circumvented if enzyme levels are correlated. In particular, if the competing catalytic domains are on the same protein but do not influence each other, the resulting bifunctional enzyme can switch sharply while remaining coherent. In the mammalian liver, the switch between glycolysis and gluconeogenesis is regulated by the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). We suggest that bifunctionality of PFK-2/FBPase-2 complements the metabolic zonation of the liver by ensuring coherent switching in response to insulin and glucagon.

Adapting glycolysis to cancer cell proliferation: the MAPK pathway focuses on PFKFB3.

Besides the necessary changes in the expression of cell cycle-related proteins, cancer cells undergo a profound series of metabolic adaptations focused to satisfy their excessive demand for biomass. An essential metabolic transformation of these cells is increased glycolysis, which is currently the focus of anticancer therapies. Several key players have been identified, so far, that adapt glycolysis to allow an increased proliferation in cancer. In this issue of the Biochemical Journal, Novellasdemunt and colleagues elegantly identify a novel mechanism by which MK2 [MAPK (mitogen-activated protein kinase)-activated protein kinase 2], a key component of the MAPK pathway, up-regulates glycolysis in response to stress in cancer cells. The authors found that, by phosphorylating specific substrate residues, MK2 promotes both increased the gene transcription and allosteric activation of PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3), a key glycolysis-promoting enzyme. These results reveal a novel pathway through which MK2 co-ordinates metabolic adaptation to cell proliferation in cancer and highlight PFKFB3 as a potential therapeutic target in this devastating disease.

PFKFB3 activation in cancer cells by the p38/MK2 pathway in response to stress stimuli.

PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) catalyses the synthesis and degradation of Fru-2,6-P2 (fructose 2,6-bisphosphate), a key modulator of glycolysis and gluconeogenesis. The PFKFB3 gene is involved in cell proliferation owing to its role in carbohydrate metabolism. In the present study we analysed the mechanism of regulation of PFKFB3 as an immediate early gene controlled by stress stimuli that activates the p38/MK2 [MAPK (mitogen-activated protein kinase)-activated protein kinase 2] pathway. We report that exposure of HeLa and T98G cells to different stress stimuli (NaCl, H2O2, UV radiation and anisomycin) leads to a rapid increase (15-30 min) in PFKFB3 mRNA levels. The use of specific inhibitors in combination with MK2-deficient cells implicate control by the protein kinase MK2. Transient transfection of HeLa cells with deleted gene promoter constructs allowed us to identify an SRE (serum-response element) to which SRF (serum-response factor) binds and thus transactivates PFKFB3 gene transcription. Direct binding of phospho-SRF to the SRE sequence (-918 nt) was confirmed by ChIP (chromatin immunoprecipiation) assays. Moreover, PFKFB3 isoenzyme phosphorylation at Ser461 by MK2 increases PFK-2 activity. Taken together, the results of the present study suggest a multimodal mechanism of stress stimuli affecting PFKFB3 transcriptional regulation and kinase activation by protein phosphorylation, resulting in an increase in Fru-2,6-P2 concentration and stimulation of glycolysis in cancer cells.

Observation of solvent penetration during cold denaturation of E. coli phosphofructokinase-2.

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N2 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner.

Expanded monomeric intermediate upon cold and heat unfolding of phosphofructokinase-2 from Escherichia coli.

Folding studies have been focused mainly on small, single-domain proteins or isolated single domains of larger proteins. However, most of the proteins present in biological systems are composed of multiple domains, and to date, the principles that underlie its folding remain elusive. The unfolding of Pfk-2 induced by GdnHCl has been described by highly cooperative three-state equilibrium (N(2)↔2I↔2U). This is characterized by a strong coupling between the subunits' tertiary structure and the integrity of the dimer interface because "I" represents an unstructured and expanded monomeric intermediate. Here we report that cold and heat unfolding of Pfk-2 resembles the N(2)↔2I step of chemically induced unfolding. Moreover, cold unfolding appears to be as cooperative as that induced chemically and even more so than its heat-unfolding counterpart. Because Pfk-2 is a large homodimer of 66 kDa with a complex topology consisting of well-defined domains, these results are somewhat unexpected considering that cold unfolding has been described as a special kind of perturbation that decouples the cooperative unfolding of several proteins.

The crystal complex of phosphofructokinase-2 of Escherichia coli with fructose-6-phosphate: kinetic and structural analysis of the allosteric ATP inhibition.

Substrate inhibition by ATP is a regulatory feature of the phosphofructokinases isoenzymes from Escherichia coli (Pfk-1 and Pfk-2). Under gluconeogenic conditions, the loss of this regulation in Pfk-2 causes substrate cycling of fructose-6-phosphate (fructose-6-P) and futile consumption of ATP delaying growth. In the present work, we have broached the mechanism of ATP-induced inhibition of Pfk-2 from both structural and kinetic perspectives. The crystal structure of Pfk-2 in complex with fructose-6-P is reported to a resolution of 2 Å. The comparison of this structure with the previously reported inhibited form of the enzyme suggests a negative interplay between fructose-6-P binding and allosteric binding of MgATP. Initial velocity experiments show a linear increase of the apparent K(0.5) for fructose-6-P and a decrease in the apparent k(cat) as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (n(H) of approximately 2). Differences and resemblances in the patterns of fructose-6-P binding and the mechanism of inhibition are discussed for Pfk-1 and Pfk-2, as an example of evolutionary convergence, because these enzymes do not share a common ancestor.

Molecular basis of the fructose-2,6-bisphosphatase reaction of PFKFB3: transition state and the C-terminal function.

The molecular basis of fructose-2,6-bisphosphatase (F-2,6-P(2)ase) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) was investigated using the crystal structures of the human inducible form (PFKFB3) in a phospho-enzyme intermediate state (PFKFB3-P•F-6-P), in a transition state-analogous complex (PFKFB3•AlF(4)), and in a complex with pyrophosphate (PFKFB3•PP(i)) at resolutions of 2.45, 2.2, and 2.3 Å, respectively. Trapping the PFKFB3-P•F-6-P intermediate was achieved by flash cooling the crystal during the reaction, and the PFKFB3•AlF(4) and PFKFB3•PP(i) complexes were obtained by soaking. The PFKFB3•AlF(4) and PFKFB3•PP(i) complexes resulted in removing F-6-P from the catalytic pocket. With these structures, the structures of the Michaelis complex and the transition state were extrapolated. For both the PFKFB3-P formation and break down, the phosphoryl donor and the acceptor are located within ~5.1 Å, and the pivotal point 2-P is on the same line, suggesting an "in-line" transfer with a direct inversion of phosphate configuration. The geometry suggests that NE2 of His253 undergoes a nucleophilic attack to form a covalent N-P bond, breaking the 2O-P bond in the substrate. The resulting high reactivity of the leaving group, 2O of F-6-P, is neutralized by a proton donated by Glu322. Negative charges on the equatorial oxygen of the transient bipyramidal phosphorane formed during the transfer are stabilized by Arg252, His387, and Asn259. The C-terminal domain (residues 440-446) was rearranged in PFKFB3•PP(i), implying that this domain plays a critical role in binding of substrate to and release of product from the F-2,6-P(2) ase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction.

Divalent metal cation requirements of phosphofructokinase-2 from E. coli. Evidence for a high affinity binding site for Mn2+.

The reaction catalyzed by E. coli Pfk-2 presents a dual-cation requirement. In addition to that chelated by the nucleotide substrate, an activating cation is required to obtain full activity of the enzyme. Only Mn(2+) and Mg(2+) can fulfill this role binding to the same activating site but the affinity for Mn(2+) is 13-fold higher compared to that of Mg(2+). The role of the E190 residue, present in the highly conserved motif NXXE involved in Mg(2+) binding, is also evaluated in this behavior. The E190Q mutation drastically diminishes the kinetic affinity of this site for both cations. However, binding studies of free Mn(2+) and metal-Mant-ATP complex through EPR and FRET experiments between the ATP analog and Trp88, demonstrated that Mn(2+) as well as the metal-nucleotide complex bind with the same affinity to the wild type and E190Q mutant Pfk-2. These results suggest that this residue exert its role mainly kinetically, probably stabilizing the transition state and that the geometry of metal binding to E190 residue may be crucial to determine the catalytic competence.

Investigating combinatorial approaches in virtual screening on human inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3): a case study for small molecule kinases.

Efforts toward improving the predictiveness in tier-based approaches to virtual screening (VS) have mainly focused on protein kinases. Despite their significance as drug targets, small molecule kinases have been rarely tested with these approaches. In this paper, we investigate the efficacy of a pharmacophore screening-combined structure-based docking approach on the human inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, an emerging target for cancer chemotherapy. Six out of a total 1364 compounds from NCI's Diversity Set II were selected as true actives via throughput screening. Using a database constructed from these compounds, five programs were tested for structure-based docking (SBD) performance, the MOE of which showed the highest enrichments and second highest screening rates. Separately, using the same database, pharmacophore screening was performed, reducing 1364 compounds to 287 with no loss in true actives, yielding an enrichment of 4.75. When SBD was retested with the pharmacophore filtered database, 4 of the 5 SBD programs showed significant improvements to enrichment rates at only 2.5% of the database, with a 7-fold decrease in an average VS time. Our results altogether suggest that combinatorial approaches of VS technologies are easily applicable to small molecule kinases and, moreover, that such methods can decrease the variability associated with single-method SBD approaches.

Ribokinase family evolution and the role of conserved residues at the active site of the PfkB subfamily representative, Pfk-2 from Escherichia coli.

Phosphofructokinase-2 (Pfk-2) belongs to the ribokinase family and catalyzes the ATP-dependent phosphorylation of fructose-6-phosphate, showing allosteric inhibition by a second ATP molecule. Several structures have been deposited on the PDB for this family of enzymes. A structure-based multiple sequence alignment of a non-redundant set of these proteins was used to infer phylogenetic relationships between family members with different specificities and to dissect between globally conserved positions and those common to phosphosugar kinases. We propose that phosphosugar kinases appeared early in the evolution of the ribokinase family. Also, we identified two conserved sequence motifs: the TR motif, not described previously, present in phosphosugar kinases but not in other members of the ribokinase family, and the globally conserved GXGD motif. Site-directed mutagenesis of R90 and D256 present in these motifs, indicate that R90 participates in the binding of the phosphorylated substrate and that D256 is involved in the phosphoryl transfer mechanism.

Structure-based drug design: from nucleic acid to membrane protein targets.

The in silico methods for drug discovery are becoming increasingly powerful and useful. That, in combination with increasing computer processor power, in our case using a novel distributed computing grid, has enabled us to greatly enhance our virtual screening efforts. Herein we review some of these efforts using both receptor and ligand-based virtual screening, with the goal of finding new anti-cancer agents. In particular, nucleic acids are a neglected set of targets, especially the different morphologies of duplex, triplex, and quadruplex DNA, many of which have increasing biological relevance. We also review examples of molecular modeling to understand receptors and using virtual screening against G-protein coupled receptor membrane proteins.

Small-molecule inhibition of 6-phosphofructo-2-kinase activity suppresses glycolytic flux and tumor growth.

6-phosphofructo-1-kinase, a rate-limiting enzyme of glycolysis, is activated in neoplastic cells by fructose-2,6-bisphosphate (Fru-2,6-BP), a product of four 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes (PFKFB1-4). The inducible PFKFB3 isozyme is constitutively expressed by neoplastic cells and required for the high glycolytic rate and anchorage-independent growth of ras-transformed cells. We report herein the computational identification of a small-molecule inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), which suppresses glycolytic flux and is cytostatic to neoplastic cells. 3PO inhibits recombinant PFKFB3 activity, suppresses glucose uptake, and decreases the intracellular concentration of Fru-2,6-BP, lactate, ATP, NAD+, and NADH. 3PO markedly attenuates the proliferation of several human malignant hematopoietic and adenocarcinoma cell lines (IC50, 1.4-24 micromol/L) and is selectively cytostatic to ras-transformed human bronchial epithelial cells relative to normal human bronchial epithelial cells. The PFKFB3 enzyme is an essential molecular target of 3PO because transformed cells are rendered resistant to 3PO by ectopic expression of PFKFB3 and sensitive to 3PO by heterozygotic genomic deletion of PFKFB3. Importantly, i.p. administration of 3PO (0.07 mg/g) to tumor-bearing mice markedly reduces the intracellular concentration of Fru-2,6-BP, glucose uptake, and growth of established tumors in vivo. Taken together, these data support the clinical development of 3PO and other PFKFB3 inhibitors as chemotherapeutic agents.

Tuning PFKFB3 Bisphosphatase Activity Through Allosteric Interference.

The human inducible phospho-fructokinase bisphosphatase isoform 3, PFKFB3, is a crucial regulatory node in the cellular metabolism. The enzyme is an important modulator regulating the intracellular fructose-2,6-bisphosphate level. PFKFB3 is a bifunctional enzyme with an exceptionally high kinase to phosphatase ratio around 740:1. Its kinase activity can be directly inhibited by small molecules acting directly on the kinase active site. On the other hand, here we propose an innovative and indirect strategy for the modulation of PFKFB3 activity, achieved through allosteric bisphosphatase activation. A library of small peptides targeting an allosteric site was discovered and synthesized. The binding affinity was evaluated by microscale thermophoresis (MST). Furthermore, a LC-MS/MS analytical method for assessing the bisphosphatase activity of PFKFB3 was developed. The new method was applied for measuring the activation on bisphosphatase activity with the PFKFB3-binding peptides. The molecular mechanical connection between the newly discovered allosteric site to the bisphosphatase activity was also investigated using both experimental and computational methods.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase and small chemical activators affect enzyme activity of activating glucokinase mutants by distinct mechanisms.

Glucokinase (GK), a monomeric glucose-phosphorylating enzyme characterised by high structural flexibility, acts as a glucose sensor in pancreatic beta cells and liver. Pharmaceutical efforts to control the enzyme are hampered by an incomplete understanding of GK regulation. We investigated GK characteristics of wild-type and activating S64Y and G68V mutant proteins in the presence of various combinations of the synthetic activators RO-28-1675 and compound A, the endogenous activator fructose-2,6-bisphosphatase (FBPase-2), and the inhibitor mannoheptulose. S64Y impedes formation of a turn structure that is characteristic for the inactive enzyme conformation, and complex formation with compound A induces collision with the large domain. G68V evokes close contact of connecting region I and helix α13 with RO-28-1675 and compound A. Both mutants showed higher activity than the wild-type at low glucose and were susceptible to further activation by FBPase-2 and RO-28-1675, alone and additively. G68V was less active than S64Y, but was activatable by compound A. In contrast, compound A inhibited S64Y, and this effect was even more pronounced in combination with mannoheptulose. Mutant and wild-type GK showed comparable thermal stability and intracellular lifetimes. A GK-6-phosphofructo-2-kinase (PFK-2)/FBPase-2 complex predicted by in silico protein-protein docking demonstrated possible binding of the FBPase-2 domain near the active site of GK. In summary, activating mutations within the allosteric site of GK do not preclude binding of chemical activators (GKAs), but can alter their action into inhibition. Our postulated GK-PFK-2/FBPase-2 complex represents the endogenous principle of activation by substrate channelling which permits binding of other small molecules and proteins.